Abstract The information stored in an organism's genome directs its development and behavior, but how that occurs is only beginning to be understood. Knowledge of what genes are used in each cell at every stage of development would be a significant step toward a comprehensive understanding. In the past grant period, we have developed technology in C. elegans that automatically assigns gene expression to individual cells at high temporal resolution up to the last round of embryonic cell division. The technology tracks the known lineage in confocal 3D movies of embryos with a ubiquitous, nuclear fluorescent label and exploits the lineage to assign expression of a second fluorescent reporter to particular cells. We have begun to apply this technology to the systematic elucidation of the expression patterns of the embryonically expressed transcription factors. In the coming grant period, we propose advances to the technology that will increase the accuracy of the lineage determination and extend it through the last round of cell division. In turn, we propose to apply this improved technology to complete the catalog of expression patterns for the embryonic transcription factors and as time permits extend this study to other key developmental genes such as receptor mediated protein kinases. We will continue to develop tools to view and analyze the data, allowing us to infer relationships between regulatory genes. Combined with emerging data sets, such as the sites of transcription factor binding through ChIP-seq studies and the phenotypes of deletion mutants for these genes, we expect that strong predictions will begin to emerge from these analyses. We will extend our technology to facilitate the use of expression patterns to validate such inferred relationships. Finally, we will develop web-based displays and tools that will permit access to our data as well as make our analytical tools available to the scientific community. These studies of gene expression patterns in C. elegans will not only provide insights into the role of these genes in development, but will suggest roles of homologous genes in human health and disease.